Rheumatoid arthritis (RA) is a chronic disease, characterized primarily by inflammation of the lining (synovium), of the joints, which can lead to long-term joint damage, resulting in chronic pain, loss of function, and disability. RA is an autoimmune disease that affects 1% of the U.S. population (2.1 million Americans), with a significantly higher occurrence among women than men. In RA, the membranes or tissues (synovial membranes) lining the joints become inflamed (synovitis). Over time, the inflammation may destroy the joint tissues, leading to disability. Because RA can affect multiple organs of the body, rheumatoid arthritis is referred to as a systemic illness. The onset of RA is usually in middle age, but frequently occurs in one's 20s and 30s.
RA progresses in three stages. The first stage involves the swelling of the lining of the joints, causing pain, warmth, stiffness, redness, and swelling around the joint. The second stage involves the thickening of the lining of the joints. During the third stage, the inflamed cells release enzymes that may digest bone and cartilage, often causing the involved joint to lose its shape and alignment, and leading to increased pain and loss of movement. Rheumatoid arthritis can start in any joint, but it most commonly begins in the smaller joints of the fingers, hands and wrists. Joint involvement is usually symmetrical, meaning that if a joint hurts on the left hand, the same joint will hurt on the right hand. In general, more joint erosion indicates more severe disease activity.
Other RA-associated symptoms include fatigue, stiffness, weakness, flu-like symptoms, including a low-grade fever, pain associated with prolonged sitting, the occurrence of flares of disease activity followed by remission or disease inactivity, rheumatoid nodules (lumps of tissue under the skin), muscle pain, loss of appetite, depression, weight loss, anemia, cold or sweaty hands and feet, and involvement of the glands around the eyes and mouth leading to decreased production of tears and saliva (Sjögren's syndrome). Advanced changes include damage to cartilage, tendons, ligaments and bone, which causes deformity and instability in the joints. The damage can lead to limited range of motion, resulting in daily tasks (grasping a fork, combing hair, buttoning a shirt) becoming more difficult. Skin ulcers and a general decline in health may also occur.
At present, RA is a chronic disease that can be controlled, but not cured. The goal of treatments is relief of symptoms and preventing the disease from worsening. Current methods of treatment of RA are focused on relieving pain, reducing inflammation, stopping or slowing joint damage, and improving a person's ability to function.
Nonsteroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, indomethacin, and COX-2 inhibitors such as valdecoxib and celecoxib, can be used to reduce inflammation and relieve pain. However, NSAIDs do not control the disease or inhibit disease progression. Analgesic drugs, including acetaminophen, propoxyphene, meperidine, and morphine, may be used to relieve pain, but they do not reduce inflammation, control the disease, or inhibit disease progression. Glucocorticoids or prednisone may be used at low maintenance doses to slow joint damage due to inflammation, but long-term use is not recommended. Disease-modifying anti-rheumatic drugs (DMARDs) are used to control the progression of RA and to try to prevent joint deterioration and disability. These anti-rheumatic drugs are often given in combination with other anti-rheumatic drugs or with other medications, such as NSAIDs or prednisone. Examples of DMARDs prescribed for rheumatoid arthritis include antimalarial medications, such as hydroxychloroquine or chloroquine, methotrexate, sulfasalazine, and oral gold. Biologic response modifiers, which directly modify the immune system by inhibiting cytokines, are also used to inhibit inflammation and RA progression. Examples of biologic response modifiers include etanercept, infliximab, adalimumab and anakinra. Some of the DMARDs and biologic response modifiers can take up to six months to work, and many have serious side effects. Protein-A immunoadsorption therapy is also used to inhibit inflammation by filtering the blood to remove antibodies and immune complexes that promote inflammation; however, this therapy offers only temporary relief of RA-associated inflammation.
Multiple sclerosis (MS) is also a chronic and potentially debilitating disease. MS affects the central nervous system (CNS), which is made up of the brain and spinal cord. MS is widely believed to be an autoimmune disease in which the body generates antibodies and white blood cells against cells that produce the myelin sheath. The myelin sheath is the fatty substance that insulates nerve fibers in the CNS, and an onslaught of the myelin sheath by such antibodies or white blood cells leads to inflammation, injury, and detachment of the myelin sheath from the nerve fiber (called demyelination). Demyelination can ultimately lead to injury of the nerves that the myelin sheath originally surrounded. Demyelination can lead to multiple areas of scarring (called sclerosis) in the CNS. Eventually, the damage induced by demyelination can slow or block nerve signals that control muscle coordination, strength, sensation, and vision. MS affects an estimated 300,000 people in the U.S. and is predicted to affect more than 1 million people worldwide. Most people first experience MS symptoms between the ages of 20 and 40 years.
MS symptoms vary depending on the location of the sclerosis and the affected nerve fibers. MS-associated symptoms may include: numbness in one or more limbs (typically occurring on one side of the body at a time, or on the bottom half of the body), partial or complete loss of vision (usually in one eye at a time, and often accompanied by pain during eye movement), double vision or blurring of vision, electric shock sensations that occur with certain head movements, tremors, lack of coordination or unsteady gait, fatigue, dizziness, muscle stiffness or spasticity, slurred speech, paralysis, problems with bladder, bowel, or sexual function, and mental changes, such as forgetfulness or difficulties with concentration.
Current treatments for MS include beta interferons (interferon beta-1b and interferon beta-1a, which help fight viral infection and regulate the immune system; these medications reduce but do not eliminate flare-ups. Beta interferons do not reverse damage, and have not been proven to significantly alter the long-term development of permanent disability. Furthermore, some individuals develop antibodies against beta interferons, which may make them less effective. Glatiramer is an alternative to beta interferons used to treat MS and it is believed to block the immune system's attack on myelin. Natalizumab is an antibody drug that blocks the attachment of immune cells to brain blood vessels, which is required for immune cells to enter the brain, thereby reducing the inflammatory action of immune cells on the nerve cells of the brain. However, natalizumab has been associated with a rare, often fatal, brain disorder called progressive multifocal leukoencephalopathy and is thus considered a high risk treatment option. The chemotherapy drug mitoxantrone has been approved for the treatment of certain aggressive forms of MS. However, due to serious side effects, such as heart damage, mitoxantrone is not used for long-term MS treatment, and it is reserved for individuals with severe attacks or rapidly advancing disease who fail to respond to other treatments.
Thus, a need exists for new, therapeutically effective drugs for the treatment of RA. Furthermore, none of the available MS therapies provide an ideal MS treatment option. Thus, there also remains a need in the art for the identification of additional agents with a demonstrated ability to treat MS in vivo. The colony stimulating factor 1 receptor (referred to herein as CSF1R; also referred to in the art as FMS, FIM2, C-FMS, and CD115) is a single-pass transmembrane receptor with an N-terminal extracellular domain (ECD) and a C-terminal intracellular domain with tyrosine kinase activity. Ligand binding of the colony stimulating factor 1 ligand (referred to herein as CSF1; also referred to in the art as MCSF and MGC31930); or the interleukin 34 ligand (referred to herein as IL34; also referred to in the art as C16orf77 and MGC34647) to CSF1R leads to receptor dimerization, upregulation of CSF1R protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream signaling events. Both CSF1 and IL34 stimulate monocyte survival, proliferation, and differentiation into macrophages. However, IL34 was discovered recently, and its overall functions have not been fully established.
Disregulation of CSF1R activity may result in an imbalance in the levels and/or activities of macrophage cell populations, which may lead to autoimmune disease and RA-associated pathology. Based on their known and suspected contributions to human autoimmune disease, both CSF1R and CSF1 have been identified as potential therapeutic targets for RA. Indeed, CSF1R and CSF1 antagonists, such as antibodies directed against CSF1R or CSF1 (see e.g., Kitaura et al., The Journal of Clinical Investigation 115(12):3418-3427 (2005), and WO 2007/081879), antisense- and siRNA-mediated silencing of CSF1R or CSF1 expression (see e.g., WO 2007/081879), soluble forms of the CSF1R ECD (see e.g., WO 2007/081879), and small molecule inhibitors of CSF1R tyrosine kinase activity (see e.g., Irvine et al., The FASEB Journal 20: 1315-1326 (2006), and Ohno et al., Clinical Immunology 38: 283-291 (2008)) and inhibitors of CSF1 (see e.g., WO 2007/081879), have been proposed for targeting RA. Despite the proposed utility of such CSF1R and CSF1 antagonists, there remains a need in the art for the identification of additional agents with a demonstrated ability to treat RA in vivo.
The inventors have also found that certain of the CSF1R ECD fusion molecules exhibit improved properties, including improvements to therapeutically relevant properties. For example, the inventors have found that expression of CSF1R ECD fusion molecules in CHO cells results in more highly sialylated CSF1R ECD fusion molecules, which are more stable than such fusion molecules produced in 293-6E cells. Also, the inventors have found that a CSF1R ECD fusion molecule wherein the CSF1R ECD has the amino acid sequence of SEQ ID NO.:2 (amino acids 20-506 of the human CSF1R protein) binds the CSF1R ligands CSF1 and IL34 more tightly and more effectively inhibits monocyte growth in an in vitro assay than a full-length CSF1R ECD fusion molecule wherein the CSF1R ECD has the amino acid sequence of SEQ ID NO.:1 (amino acids 20-512 of the human CSF1R protein). Thus, this CSF1R ECD fusion molecule provides a particularly attractive therapeutic molecule.
The inventors have also found that CSF1R ECD fusion molecules are effective in treating MS and RA in in vivo models (See Examples 8, 9, and 13). Furthermore, CSF1R ECD fusion molecules are also effective to deplete particular classes of monocytes from peripheral blood and spleen, respectively, as shown in Examples 7 and 14. Accordingly, some embodiments of the application include methods and compositions for treating RA or MS. Other embodiments of the invention further include methods and compositions for depleting peripheral blood monocytes, inhibiting monocyte viability, and inhibiting CSF1- and/or IL34-stimulated monocyte proliferation. Furthermore, in certain embodiments, CSF1R ECD fusion proteins of the invention may be used for treating other inflammatory conditions such as psoriasis, SLE (lupus), COPD, atopic dermatitis, and atherosclerosis, as well as macrophage activation syndrome and histiocytosis X.
CSF1R ECD fusion molecule of the invention include, for example, a CSF1R ECD fusion molecule and one or more fusion partners, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises SEQ ID NO.:2 (corresponding to human CSF1R ECD residues 1-506) and excludes the last six C-terminal amino acid residues of SEQ ID NO.:1 (corresponding to human CSF1R ECD residues 507-512). In such fusion molecules, any amino acid residues that follow the C-terminal residue of SEQ ID NO:2 do not begin with the amino acid sequence of residues 507-512 of SEQ ID NO:1 (THPPDE). Such fusion molecules may of course include the amino acid sequence THPPDE anywhere else in the amino acid sequence. In some such embodiments, the CSF1R ECD consists of SEQ ID NO:2.
A CSF1R ECD fusion molecule wherein the amino acid sequence of the CSF1R ECD corresponds to SEQ ID NO.:2 showed higher affinity for CSF1 and IL34 ligands than the CSF1R ECD fusion molecule wherein the amino acid sequence of the CSF1R ECD corresponds to SEQ ID NO.:1. A CSF1R ECD fusion molecule wherein the amino acid sequence of the CSF1R ECD corresponds to SEQ ID NO.:2 also inhibited monocyte viability and CSF1- and IL34-stimulated proliferation of human monocytes better than the CSF1R ECD fusion molecule wherein the amino acid sequence of the CSF1R ECD corresponds to SEQ ID NO.:1. Thus, in another aspect of the invention, the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of the hCSF1R.506-Fc fusion molecule described above (SEQ ID NO.:6).
The one or more fusion partners in any of the embodiments described previously includes, but is not limited to, an Fc, albumin, or polyethylene glycol, or both an FC and polyethylene glycol. In some embodiments, the fusion molecule comprises a linker between the CSF1R ECD and one or more fusion partners. In some such embodiments, the linker is a peptide consisting of the amino acid sequence glycine-serine. For example, in some embodiments, the CSF1R ECD fusion molecule comprises a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6.
In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells. The present invention also provides pharmaceutical compositions comprising the CSF1R ECD fusion molecules of the invention and a pharmaceutically acceptable carrier.
The present invention further provides a polynucleotide comprising a nucleic acid sequence that encodes any one of the above described CSF1R ECD fusion molecules of the invention. In some embodiments, the amino acid sequence encoded by the polynucleotide of the invention comprises a signal peptide amino acid sequence. In some embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO:2, wherein the amino acid sequence excludes the six C-terminal residues of SEQ ID NO:1. In other embodiments, the polynucleotide encodes an amino acid sequence comprising SEQ ID NO:6 plus a signal peptide amino acid sequence, such as, for example, SEQ ID NO:16. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 39. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 40. Another aspect of the invention provides an expression vector comprising the polynucleotide described above.
It has also been found that the CSF1R ECD fusion molecule is more highly sialylated when produced from CHO cells compared to the fusion molecule produced from other cells, such as 293-6E cells. Thus, the present invention also provides a CHO cell comprising an expression vector encoding the CSF1R ECD fusion molecule and a method of producing the CSF1R ECD fusion molecule of the invention from a CHO cell. For example, in some embodiments, the method comprises: (a) culturing a CHO cell comprising the polynucleotide of any one of the above described CSF1R ECD fusion molecules in conditions such that the CSF1R ECD fusion molecule is expressed; and (b) recovering the CSF1R ECD fusion molecule. The invention further includes this method with the step of fusing polyethylene glycol to the CSF1R ECD fusion molecule. The present invention further provides a method for producing glycosylated and sialylated CSF1R ECD fusion molecules. For example, in some embodiments, the CHO cell comprises a vector comprising a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO:6 plus a signal peptide amino acid sequence, such as, for example, SEQ ID NO:16. In some embodiments, the CHO cell comprises a vector comprising a polynucleotide sequence that comprises the sequence of SEQ ID NO: 39. In some embodiments, the CHO cell comprises a vector comprising a polynucleotide sequence that comprises the sequence of SEQ ID NO: 40.
Methods of the invention also comprise administering to a patient a therapeutically effective amount of a CSF1R ECD fusion molecule, wherein the fusion molecule comprises a CSF1R ECD and one or more fusion partners. The invention provides, for example, a method of treating multiple sclerosis, a method of treating rheumatoid arthritis, or a method of depleting peripheral blood monocytes in a patient comprising administering to the patient a therapeutically effective amount of a CSF1R ECD fusion molecule. In some embodiments of those methods, the CSF1R ECD of the CSF1R ECD fusion molecule comprises the full-length human CSF1R ECD (hCSF1R.512; SEQ ID NO.:1). In other embodiments, the CSF1R ECD fusion molecule comprises SEQ ID NO.:2 (corresponding to human CSF1R ECD residues 1-506) and excludes the last six C-terminal amino acid residues of SEQ ID NO.:1 (corresponding to human CSF1R ECD residues 507-512). In some such embodiments, the CSF1R ECD consists of SEQ ID NO:2. In a further aspect, the CSF1R ECD of the CSF1R ECD fusion molecule comprises the full-length CSF1R ECD of SEQ ID NO.:1, but excludes the last C-terminal amino acid residue of SEQ ID NO.:1 (referred to herein as CSF1R.511; SEQ ID NO.:26). In some such embodiments, the CSF1R ECD consists of SEQ ID NO:26 or SEQ ID NO:1.
The one or more fusion partners in any of the embodiments described previously includes, but is not limited to, an Fc, albumin, or polyethylene glycol, or both an FC and polyethylene glycol. In some embodiments, the fusion molecule comprises a linker between the CSF1R ECD and the fusion partner. In some such embodiments, the linker is a peptide consisting of the amino acid sequence glycine-serine. For example, in some embodiments, the CSF1R ECD fusion molecule comprises a CSF1R ECD, an Fc, and polyethylene glycol, wherein the amino acid sequence of the CSF1R ECD fusion molecule comprises or consists of SEQ ID NO.:6.
In some embodiments, the CSF1R ECD comprises a signal peptide. In some embodiments, the fusion molecule is glycosylated and/or sialylated. In some embodiments, the polypeptide portion of the fusion molecule is expressed in Chinese hamster ovary (CHO) cells.